2 research outputs found
De novo backbone and sequence design of an idealized α/β-barrel protein: evidence of stable tertiary structure
We have designed, synthesized, and characterized a 216 amino acid residue
sequence encoding a putative idealized α/β-barrel protein. The
design was elaborated in two steps. First, the idealized backbone was
defined with geometric parameters representing our target fold: a central
eight parallel-stranded β-sheet surrounded by eight parallel α-helices,
connected together with short structural turns on both sides of the barrel.
An automated sequence selection algorithm, based on the dead-end elimination
theorem, was used to find the optimal amino acid sequence fitting
the target structure. A synthetic gene coding for the designed sequence
was constructed and the recombinant artificial protein was expressed in
bacteria, purified and characterized. Far-UV CD spectra with prominent
bands at 222 nm and 208 nm revealed the presence of α-helix secondary
structures (50%) in fairly good agreement with the model. A pronounced
absorption band in the near-UV CD region, arising from immobilized aromatic
side-chains, showed that the artificial protein is folded in solution.
Chemical unfolding monitored by tryptophan fluorescence revealed a
conformational stability (ΔGH_2O) of 35 kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T_m of 65 °C. Moreover, the artificial protein did not exhibit any affinity
for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic
acid (ANS), providing additional evidence that the artificial barrel is not
in the molten globule state, contrary to previously designed artificial a/
b-barrels. Finally, ^1H NMR spectra of the folded and unfolded proteins
provided evidence for specific interactions in the folded protein. Taken
together, the results indicate that the de novo designed α/β-barrel protein
adopts a stable three-dimensional structure in solution. These encouraging
results show that de novo design of an idealized protein structure of
more than 200 amino acid residues is now possible, from construction of
a particular backbone conformation to determination of an amino acid
sequence with an automated sequence selection algorithm